Adhesion of mycoplasma pneumoniae and mycoplasma hominus to sulfatide

ABSTRACT

The invention is a carbohydrate receptor for Mycoplasma pneumoniae and Mycoplasma hominus and its use to detect mycoplasma in biological fluids and diseased tissue and cells. The receptor can be included in a composition having a pharmaceutically acceptable carrier. Methods are provided for purifying, detecting, or removing mycoplasma from diseased tissue or fluids. The receptor includes sulfatides, dextran sulfate, sialyloligosaccharides, and mixtures thereof.

FIELD OF THE INVENTION

The present invention relates generally to carbohydrate receptors andtheir use. Specifically, for the detection of Mycoplasma pneumoniae andMycoplasma hominus, a method for removing Mycoplasma pneumoniae andMycoplasma hominus from fluids, and for inhibiting the growth ofMycoplasma pneumoniae and Mycoplasma hominus.

BACKGROUND OF THE INVENTION

Mycoplasmas are a group of microorganisms which are intermediate in sizebetween bacteria and viruses. Included among the human Mycoplasmaspecies which to date have been characterized are Mycoplasma hoministypes 1 and 2, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasmaorale types 1 and 2, and Mycoplasma pneumoniae. The species M. hoministype 1 has been recovered from the genitourinary tract, and its frequentoccurrence in association with venereal disease, non-bacterialurethritis, cervicitis, and other inflammatory diseases of the genitaltract has been reported as well as its association with exudativepharyngitis. M. pulmonis, while not indigenous to man, has been isolatedfrom tissue cultures inoculated with specimens from leukemia patients.M. hominis type 2, which is occasionally isolated from human specimens,has been shown to be identical to a rodent mycoplasma, M. arthritidis.

The rapid identification of mycoplasmas becomes extremely important ininitiating the proper treatment of illnesses of which mycoplasmas arethe causative agent, especially because they are resistant to many ofthe antibiotics and chemotherapeutic agents used for bacterialinfections.

In recent years, it has been reported that mycoplasmas are present, ascontaminants, in tissue cultures such as are used in the metabolicstudies of cells or in the propagation of viruses. A prime contaminanthas been identified as M. hominis, type 1. The occurrence of mycoplasmain tissue cultures furnishes a potential source for an erroneousinterpretation of results, since the interpretation invariably presumesthat cultures are devoid of microbial contaminants.

Techniques and methods for isolating, identifying, and inhibiting thegrowth of mycoplasmas, particularly the human strains, therefore havebecome important in the preparation and use of tissue cultures.

Mycoplasma pneumoniae is a small prokaryotic parasite of the humanrespiratory tract and the etiologic agent of primary atypical pneumonia.This pathogen has no cell wall, and requires exogenous cholesterol forthe synthesis of plasma membrane and glucose as a carbon and energysource. In tracheal organ cultures the adhesion of viable mycoplasms tothe respiratory epithelium is essential for the initiation of infection(cf. Collier et al., Infect. Immun. 3: 694-701, 1971; Hu et al., op.cit. 11: 704-710, 1975; Hu et al. op. cit. 14: 217-224, 1976). Oncebound, M. Pneumonia does not penetrate the epithelial surface, butcauses extensive damage to the tracheal epithelium, leading tociliostasis, loss of cilia, and finally, cell death (Collier et al., inPathogenic Microplasmas, Ciba Foundation Symposium, Jan. 25 to 27, 1972,Elsevier/North-Holland, Amsterdam, p. 307-327 and Carson et al., Infect.Immun. 29: 1117-1124, 1980).

M. pneumoniae also binds in vitro to many other eucaryotic cells,including human colon carcinoma cells (WiDr), human lung fibroblasts(MRC5), HeLa cells, hamster tracheal epithelial cells, spermatozoa, anderythrocytes. Some of these studies suggest that sialylglycoproteins maybe receptors for M. pneumoniae, as treatment of the cells withneuraminidase decreases binding (Manchee et al., Br. J. Exp. Pathol. 50:66-75, 1969; Sobeslavsky et al., J. Bacgeriol. 96: 695-705, 1968; andBarile, M. F. in The Mycoplasmas, Vol. II, Tully et al., Eds., pp425-464, Academic Press, New York, 1979).

Recent studies suggest that the organism recognizes Neu-Acα2-3Galβ1-4GlcNAc sequences on erythrocytes, as both glycolipids andglycoproteins containing this structure inhibit adhesion of bacteria,cf. Loomes et al., Nature (Lond) 307: 560-563, 1984; and Loomes et al.,Infect. Immun. 47: 15-20, 1985. Other studies, however, suggest thatglycolipids are not receptors for M. pneumoniae, cf. Gabridge et al.,Infect. Immun. 25: 455-459, 1979; and Geary et al., Isr. J. Med. Sci.23: 462-468, 1987.

A number of prior art workers have provided methods for detecting andidentifying mycoplasmas. For example, Cekoric et al. in U.S. Pat. No.3,668,075 disclose a method for identifying groups of mycoplasmas basedon the fact that certain heparinoid compounds selectively inhibit thegrowth of mycoplasmas in growth media.

Makela et al. in U.S. Pat. No. 4,652,518 disclose a preparation fordetecting chlamydial infections using a lipopolysaccharide forRe-lipopolysaccharide mutants of gram-negative bacteria. Thepolysaccharide is complexed to a carrier molecule to enhanceimmunological response.

Waters et al. in U.S. Pat. No. 4,632,902 disclose a method for detectingbiological activity using a nutrient growth medium which isolatesantibiotics and other microbial growth inhibitors during culturing of amicroorganism. The growth medium contains an isolating substance whichisolates antimicrobial materials during culturing of a microorganism.The isolating substances may be ion exchange resins or non-functionaladsorbent resins.

Keller et al. in U.S. Pat. No. 4,543,328 disclose a method forseparating bacteria, fungi, and viruses from blood during extracorporealcirculation of the blood with a biocompatible adsorbent. These polymersare blood compatible, and may be polyacrylates, polymethacrylates,crosslinked polystyrenes, cellulose acetate, collodium, and nylon.

Japanese patent 55-31959 discloses a latex for diagnosis of Mycoplasmapneumoniae infectious diseases comprising a Mycoplasma pneumoniae lipidantigen bound with a suspension of latex particles. The lipid antigenmay be produced by extracting Mycoplasma fungi bodies with an organicsolvent. The Mycoplasma pneumoniae strains are cultured and the fungibodies are collected and extracted with an organic solvent. A sensitizedlatex is produced by adding a solution containing lipid antigen into asuspension where latex particles are suspended, and treating theresultant mixture for 2-4 hours. The latex sensitized with lipid antigenmay be conserved at 4° C. for longer than one hour by adding aprotecting agent such as glycine or dextran and freeze-drying.

Schiefer et al. in Soecialia Aug. 15, 1978, p. 1011, disclose thatsurface carbohydrate structures can be visualized on Mycoplasmamembranes using a cytochemical staining procedure with concanavalin Aand iron-dextran complexes. However, there is no disclosure that thisstaining can be used for diagnostic purposes.

SUMMARY OF THE INVENTION

It is an object of the present invention to advance the treatment ofMycoplasma induced disease, and more particularly to provideimprovements in the detection of Mycoplasma induced disease.

It is a further object of the present invention to provide receptorsthat mediate binding of M. pneumoniae means and M. hominus to cells.

It is another object of the present invention to inhibit the adhesion ofM. pneumoniae and M. hominus to human cells and tissues.

It is yet another object of the present invention to provide a methodfor detecting the presence of M. pneumoniae and M. hominus in biologicalfluids.

It is still another object of the present invention to provide a methodfor detecting the presence of M. pneumoniae and M. hominus in tissuecultures,

According to the present invention, dextran sulfate, but not otherpresently known sulfated or anionic polysaccharides, completely inhibitsbinding of M. pneumoniae and M. hominus to purified sulfatide. Thedextran sulfate partially inhibits adhesion of M. pneumoniae and/or M.hominus to cultured human colon adenocarcinoma cells.

The presence or absence of M. pneumoniae or M. hominus in a biologicalsample is determined according to the present invention by contractingthe sample with dextran sulfate, and then testing for the presence of M.pneumoniae or M. hominus such as by staining and thin layerchromatography.

Dextran sulfate inhibits the binding of M. pneumoniae and M. hominus toa variety of human cells containing sulfatide receptors, including cellswhich contain terminal Gal(3SO4)β1-residues. Since dextran sulfateinhibits the binding of this microorganism to cells, the administrationof dextran sulfate to a patient or host infected with this microorganismcan prevent the growth and multiplication of this microorganism byinhibiting its binding to human cells.

Because M. pneumoniae in a sample can interfere with analyses for avariety of other substances, it is often desirable to remove the M.pneumoniae from the fluid being tested prior to performing the tests.Either dextran sulfate alone, or a combination of asialyloligosaccharide, i.e. a compound containing α2-3-linked sialicacid, and dextran sulfate is adsorbed onto an insoluble carrier, and thetest fluid then contacted with this carrier. The M. pneumoniae thenadhere to the carrier, and can be effectively removed from the testfluid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of M. pneumoniae to glycolipids separated bythin layer chromatography.

FIG. 2 shows binding of M. pneumoniae to purified glycolipids.

FIG. 3 shows the energy and temperature dependent binding of M.pneumoniae to sulfatide.

FIG. 4 shows the inhibition of M. pneumoniae binding to sulfatide bydextran sulfate.

FIG. 5 shows the identification of sulfatide synthesized by WiDradenocarcinoma cells.

FIG. 6 shows M. pneumoniae binding to immobilized glycoproteins

FIG. 7 shows the effect of neuramidase treatment on M. pneumoniaebinding to immobilized glycoproteins.

FIG. 8 shows the structures of sialylated oligosaccharides on the αsubunit of human chorionic gonadotropin that mediates M. pneumoniaeadhesion.

FIG. 9 shows the inhibition of M. pneumoniae adhesion to humanadenocarcinoma cell lines (WiDr).

DETAILED DESCRIPTION OF THE INVENTION

Dextran sulfate inhibits the binding of M. pneumoniae to a variety ofhuman cells containing sulfatide receptors, including glycolipids suchas seminolipid and lactosylsulfatide , which all contain terminalGal(3SO₄)β1- residues.

According to the present invention, a virulent strain of Mycoplasmapneumoniae was metabolically labelled with [³ H]palmitate and studiedfor binding to glycolipids and to WiDr human colon adenocarcinoma cells.It was found that the organism bound strongly to sulfatide and to othersulfated glycolipids, such as seminolipid and lactosylsulfatide, whichall contain terminal Gal(3SO₄)β1-residues.

M. pneumoniae binds only weakly or not at all to many gangliosides,including the sialylneolacto-series and neutral glycolipids. Onlymetabolically active M. pneumoniae cells bind to sulfatide, as bindingis maximal in RPMI medium at 37° C., and is almost completely abolishedin nutrient-deficient medium or by keeping the cells at 4° C.

This is particularly relevant because sulfatide occurs in large amountsin human trachea, lung, and WiDr cells, and the administration ofdextran sulfate can thus inhibit binding of the M. pneumoniae to thesehuman cells.

Several purified glycoproteins, including laminin, fetuin, and humanchorionic gonadotropin, promote dose-dependent and saturable adhesion ofM. pneumoniae when adsorbed on plastic. Adhesion to the proteins isenergy dependent, as no attachment occurs in media without glucose.Adhesion to all of the proteins requires sialic acid, and only thoseproteins with α2-3-linked sialic acid are active. The α-subunit of humanchorionic gonadotropin also promotes attachment, suggesting that asimple biantennary asparagine-linked oligosaccharide is sufficient forbinding. Soluble laminin, asparagine-linked sialyloligosaccharides fromfetuin, and 3'-sialyllactose but not 6'-sialyllactose inhibit attachmentof M. pneumoniae to laminin. M. pneumoniae also bind to sulfatideadsorbed on plastic. Dextran sulfate, which inhibits M. pneumoniaebinding to sulfatide, does not inhibit attachment on laminin, and3'-sialyllactose does not inhibit binding to sulfatide, suggesting thattwo distinct receptor specificities mediate binding to these twocarbohydrate receptors. Both 3'-sialyllactose and dextran sulfatepartially inhibit M. pneumoniae adhesion to a human colon adenocarcinomacell line (WiDr) at concentrations that completely inhibit binding tolaminin or sulfatide, respectively, and in combination they inhibitbinding of M. pneumoniae to these cells by 90%. Thus, both receptorspecificities contribute to M. pneumoniae adhesion to cultured humancells.

Bovine brain sulfatide (galactosyl ceramide-I³ -sulfate), ceramidemonohexoside, ceramide trihexoside, globoside, and gangliosides GM1 andGD1a were obtained from Supelco. Lactosylceramide and glucosylceramidewere obtained from Calbiochem. Other reference gangliosides wereobtained from Bachem, Inc. Seminolipid (β-galactosylalkylacylglycerol-I³-sulfate) was isolated from bovine testes as described by Roberts et al.in Cancer Res. 48:3367-3373, 1988. Galactosyl ceramide-I6-sulfate wasprepared as described by Roberts et al. in J. Biol. Chem. 261:6972-6977, 1986, by sulfation of galactosyl ceramide. Sulfatedglucuronosylparagloboside (IV³ -[3'SO₃ GlcA]- nLcOse4Cer) was purifiedfrom human peripheral nerve as reported by Chou et al. in J. Biol. Chem.261:11717-11725, 1986. Lactosylceramide-II³ -sulfate, GM3, andsialyllactofucopentaosyl-(III)-ceramide were purified from human kidneyas described by Rauvala et al in J. Biol. Chem. 251:7517-7520, 1976, andHanfland et al., in Biochemistry 20:5310-5319, 1981.α-galactosylparagloboside (IV³ GalnLcOse4Cer) and the I-activeα-Ga121actoisooctaosylceramide were purified from rabbit erythrocytes asdescribed by Watanabe et al. in J. Biol. Chem. 254: 3221-3228, 1979.Lactoisooctaosylceramide was prepared from the latter lipid by treatmentwith coffee bean α-galactosidase. Alpha2-3-sialylparagloboside (NeuGc),α2-3-sialyllactoneohexaosylceramide, GM3 (NeuGc), and an I-activeganglioside were prepared from bovine erythrocytes according to theprocess of Watanabe et al. in J. Biol. Chem. 254:8223-8228, 1979.α2-3-sialyl-paragloboside (NeuAc) was isolated from type O humanerythrocytes as described by Ardo et al. in J. Biochem. 79: 625-632,1976.

Paragloboside and lactoneohexaosylceramide were prepared bydesialylation of the respective gangliosides with 1 M formic acid forsixty minutes at 100° C. Lacto-N-triaosylceramide was prepared bydigestion of paragloboside with bovine testes β-galactosidase.

The identities of the neolacto-series glycolipids was confirmed byimmunostaining with monoclonal antibody My-28 before and afterneuraminidase digestion. Lipids were extracted from normal human lung,trachea, and WiDr cells and separated into neutral and acidic fractionsby anion exchange chromatography on DEAE-Sepharose in the bicarbonateform. For some experiments WiDr cells were metabolically labelled with[³⁵ S]-sulfate. Cells were removed from the tissue culture flasks byremoving the medium and adding 2.5 mM EDTA in 10 mM phosphate bufferedsaline pH 7.3. After sixty minutes at 37° C., the cells were collectedby centrifugation and extracted as described above. The sulfatedglycolipids in the tissue extracts were detected by staining of thelipids separated by high performance thin layer chromatography with ¹²⁵I-von Willebrand factor.

Growth and Labelling of Organisms

Virulent M. pneumoniae strain M129, passage 5-15, were grown andmetabolically labelled with [³ H]palmitic acid, 12-17 Ci/mole, asdescribed by Chandler et al. in Infect. Immun. 37: 942, 1982. Theorganisms were passed four times through a 26 gauge needle and suspendedto approximately 10⁷ cpm/ml of degassed RPMI 1640 medium containing 1%bovine serum albumin and 25 mM HEPES, pH 7.3.

Mycoplasma Overlay Assay

M. pneumoniae were bound to glycolipids separated on thin-layerchromatograms as described in detail for other bacteria by Krivan etal., Proc. Natl. Acad. Sci. 85: 6157-6161, 1988, and Krivan et al.,Arch. Biochem. Biophys. 260:493-496, 1988. Briefly, glycolipids wereseparated by thin-layer chromatography on aluminum-backed silica gelhigh-performance plates developed with chlooform:methanol:0.25% CaCl₂ inwater (60:35:8). After chromatography, the plates were coated with 0.1%polyisobutylmethacrylate, soaked in 0.05 M Tris-HCl, pH 7.6, containing110 nM sodium chloride, 5 mM CaCl₂, 0.2 mM phenylmethane-sulfonylfluoride, and 1% bovine serum albumin (TBS-BSA) and incubated for threehours at 25° C. with 60 μl/cm² of [³ H]-labelled M. pneumoniae,approximately 10⁷ cpm/ml of RPMI-BSA. The plates were gently washed fivetimes in 0.01 M sodium phosphate, pH 7.2, containing 0.15 M sodiumchloride (PBS) to remove unbound organisms, dried, and exposed for 24hours to Ultrofilm ³ H (2208-190) high speed film.

Solid-Phase Binding Assay

The binding of M. pneumoniae to purified glycolipids immobilized inmicrotiter plates was measured as described by Krivan et al. in Proc.Natl. Acad. Sci., op. cit. Purified glycolipids were serially diluted in25 μl of methanol containing 0.1 μg each of the auxiliary lipidscholesterol and phosphatidylcholine. After the solutions were dried byevaporation, the wells were filled with TBS-BSA, emptied after one hour,rinsed with RPMI-BSA, and incubated with 25 μl of [³ H]-M. pneumoniae,approximately 10⁷ cpm/ml RPMI-BSA. After incubation for two hours at 37°C., unless otherwise stated, the wells were washed five times withsaline and bound M. pneumoniae was quantified by scintillation countingin Aquasol. For inhibition studies, various polysaccharides wereserially diluted in 25 ml of [³ H]-M. pneumoniae.

Mycoplasm adhesion to cultured cells

Adhesion of [³ H]-M. pneumoniae to cells on glass covered slips wasmeasured by a modification of a method previously described by Chandleret al., Infect. Immun., op. cit. WiDr human colon adenocarcinoma, ATCCCCL 218 was grown in Eagle's minimal essential medium with 10% fetalcalf serum in a 5% CO₂ atmosphere at 37° C. The cells were removed withtrypsin and plated on 12 mm round glass coverslips in 24-well tissueculture plates and grown for three days. Control coverslips werepreincubated in medium without cells. The coverslips were washed inserum-free medium, then incubated in RPMI-BSA for fifteen minutes. Themedium was removed and labelled M. pneumoniae suspended in 0.5 ml ofRPMI-BSA were added to each well. The plates were incubated on a rockingtable for 60 minutes at 37° C. The coverslips were washed by dipping insaline six times, and the bound radioactive bacteria were determined byscintillation counting. For inhibition studies, the inhibitors wereadded to M. pneumoniae prior to adding the bacteria to the coverslips.

Binding of M. pneumoniae to Glycolipids on Thin Layer chromatograms

Incubation of [³ H]-labelled M. pneumoniae with various glycolipidsresolved on thin layer chromatograms was used to determine thecarbohydrate binding specificity of the organism. As shown by anautoradiogram, FIG. 1A, compared with a similar thin layer platevisualized with orcinal reagent, FIG. 1B, M. pneumoniae bound avidly toauthentic sulfatide, detecting 100 ng of this glycolipid, lane C₃, andto a glycolipid with the same mobility as sulfatide in the acidic lipidfraction of human trachea, lane f. This tracheal glycolipid wasconfirmed to be sulfatide by its specific staining with ¹²⁵ I-labelledvon Willebrand factor. Sulfatide was also detected in human lung lipidsbut at lower levels than in trachea.

M. pneumoniae also bound to other sulfated glycolipids includinglactosyl sulfatide and seminolipid, which contain the same terminalGal(3SO₄)β1-residue as sulfatide, and an isomer of sulfatide in whichthe terminal sulfate is linked to the 6-position of galactose. Table 1shows the structures of interest.

M. pneumoniae also binds to high amounts of lactosylderamide and to alesser extent glucosylceramide, paragloboside, ando-galactosylparagloboside, but not to other neutral glycolipids, asshown in Table 1. No binding was detected to other acidic glycolipidsincluding α2-3-sialylparagloboside, I-active monosialylganglioside, orto the gangliosides GM3, GM2, GM1, GD1a, GS1b, and GT1b. In addition,sulfate itself is not sufficient for binding, as M. pneumoniae does notbind to high amounts of cholesterol sulfate or to sulfatedglucuronosylparagloboside, which has a terminal sulfate linked to the3-position of glucuronic acid.

To obtain the results shown in FIG. 1, glycolipids were chromatographedon aluminum-backed silica gel HPTLC plates developed inchloroform/methanol/0.25% CaCl₂ in water, 60:35:8. The plates werecoated with plastic, soaked in Tris-BSA, and incubated for three hoursat 25° C. with [³ H]-palmitate-labelled M. pneumoniae suspended in RPMI1640 containing 1% BSA and 25 mM HEPES, pH 7.3, as described above(Panel A), or sprayed with orcinol reagent to identify glycolipids(Panel B). Lane a, acidic glycolipid standards sulfatide (0.5 μg), GM3(2 μg), GM2 (2 μg), GD1a (2 μg), GD1b (2 μg), GT1b (2 μg); lane b,neutral standards galactosyl ceramide (4 μg), lactosylceramide (4 μg),globotriaosylceramide (2 μg), c2(0.5 μg). and c3 (o.1 μg); lane d,seminolipid (2 μg); lane e, cholesterol 3 -sulfate (2 μg); lane f, humantrachea acidic glycolipids from 100 mg wet weight of bovineerythrocytes; lane h, α2-3sialylparagloboside (2 μg); lane 1, I-activemonosialylganglioside from bovine erythrocytes (2 μg).

Quantitative Binding of M. pneumoniae to Immobilized Glycolipids inMicrotiter Plates

Binding of M. pneumoniae to purified glycolipids adsorbed on microtiterplates was examined to further define binding specificity. Binding tosulfatide was sensitive and dose-dependent, as shown in FIG. 2. M.pneumoniae bound weakly to lactosylceramide and paragloboside, whereasno binding was detected to cholesterol sulfate or other glycolipidstested at 10 μg per well, consistent with the data obtained from theoverlay assay.

Binding of M. pneumoniae to sulfatide is both energy and temperaturedependent, as shown in FIG. 3. At 37° C. about 0.25 μg of sulfatide wasrequired for half-maximum binding. The binding activity was about fivetimes lower at 25 C and was minimal at 4° C. M. pneumoniae also boundpoorly at 37° C. in nutrient-deficient medium (Tris-BSA without RPMI)with binding activities comparable to that obtained at 4° C., as shownin FIG. 3. These results suggest that M. pneumoniae requires energy andphysiological temperatures for maximal binding to occur.

To obtain the results shown in FIG. 2, lipids in 25 μg each of theauxiliary lipids cholesterol and phosphatidylcholine were evaporated inflat bottom wells of polyvinylchloride microtiter plates. The wells wereblocked with 1% albumin for one hour, washed twice with RPMI-BSA, andincubated at 25° C. with 25 μl of [³ H]-M. pneumoniae, approximately 105cpm. After two hours, the wells were washed five times with saline, cutfrom the plate, and bound radioactivity was quantified in ascintillation counter. In control experiments, organisms were incubatedwith auxiliary lipids only to correct for nonspecific binding, typically<1% of the total radioactivity added. M. pneumoniae binding wasdetermined in RPMI-BSA for sulfatide ( ○ ), lactosylceramide (),paragloboside (), and cholesterol sulfate, ceramide trihexoside,globoside, GM1, GM2, or GM3 ( ◯).

In FIG. 3, to demonstrate the energy and temperature dependent bindingof M. pneumoniae to sulfatide, microtiter wells were coated withsulfatide and blocked with albumin as described for FIG. 2. Binding of[³ H]-M. pneumoniae was determined in RPMI-BSA for two hours at 4° C.(), 25° C. ( ○ ), 37° C. (), and at 37° C. in BSA without RPMI (Δ).

Inhibition of M. pneumoniae Binding to Immobilized Sulfatide and WiDrMonolayers by Dextran Sulfate

Various anionic polysaccharides were tested for inhibition of M.pneumoniae binding to sulfatide adsorbed in microtiter plates, as shownin FIG. 4. Dextran sulfate at 0.4 μg/ml inhibited binding to 1 μg ofsulfatide by 50%, whereas dextran had no effect. At 100 μg/ml yeastphosphomannan, colominic acid, hyaluronate, and several sulfatedpolysaccharides such as fucoidin, heparin, and chondroitin sulfate didnot inhibit binding.

To obtain the results shown in FIG. 4, polysaccharides were seriallydiluted in 25 μl of RPMI-BSA in microtiter wells previously coated with1 μg of purified sulfatide. Binding was determined after incubation oftwo hours at 37° C. with 25 μl of [³ H]-M. pneumoniae with the indicatedconcentration of dextran ( ○ ) or dextran sulfate ().

Because virulent strains of M. pneumoniae adhere to mammalian cells,monolayers of WiDr cells were used to determine if dextran sulfateinhibits adhesion of the organism. [³ H] -labelled M. pneumoniae wasincubated with WiDr cell monolayers attached to coverslips in triplicatewith an without dextran sulfate, as shown in Table II. Dextran sulfateinhibited adhesion in all three experiments, but the degree ofinhibition varied between experiments. In each experiment, 10 μg/mldextran sulfate inhibited more than 1 μg/ml, but 100 μg/ml dextransulfate caused no further inhibition. Thus, maximal inhibition wasobtained with approximately 10 μg/ml of dextran sulfate. Similar resultswere obtained with MRC5 lung fibroblasts where in three experiments amean of 47% of M. pneumoniae adhesion was inhibited by dextran sulfate.

                                      TABLE I                                     __________________________________________________________________________    Glycolipids tested for ability to bind M. pneumoniae                          Name.sup.a      Structure                                Binding.sup.b        __________________________________________________________________________    Sulfatide       Gal(3SO.sub.4)β11Cer                +++                  Sulfatide       Gal(6SO.sub.4)β11Cer                +++                  Lactosylsulfatide                                                                             Gal(3SO.sub.4)β14Glcβ11Cer     +++                  Seminolipid     Gal(3SO.sub.4)β13alkylacylglycerol  +++                  Glucosylcer (CMH)                                                                             Glcβ11Cer                           +                    Lactosylcer (CDH)                                                                             Galβ14Glcβ11Cer                ++                   Lacto- .sub.--N-triaosylcer                                                                   GlcNAcβ13Galβ14Glcβ11Cer  +                    Paragloboside   Galβ14GlcNAcβ13Galβ14Glcβ11Cer                                                                     +                    α-Galactosylparagloboside                                                               Galα13Galβ14GlcNAcβ13Galβ14Glcβ1                    Cer                                      +                    Galactosylcer (CMH)                                                                           Galβ11Cer                           -                    SO.sub.4 -Glucuronosylparagloboside                                                           GlcA(3SO.sub.4)β13Galβ14GlcNAcβ13Galβ1                    Glcβ11Cer                           -                    Trihexosylcer (CTH)                                                                           Galα14Galβ14Glcβ11Cer    -                    Asialo GM2      GalNAcβ14Galβ14Glcβ11Cer  -                    Globoside (GLA) GalNAcβ13Galα14Galβ14Glcβ11Cer                                                                    -                    Asialo GM1      Galβ1 3GalNacβ14Galβ14Glcβ11Cer                                                                    -                    GM3             NeuAcα23Galβ14Glcβ11Cer  -                    GM3 (NeuGc)     NeuGcα23Galβ14Glcβ11Cer  -                    GM2             GalNAcβ14[NeuAcα23]Galβ14Glcβ11Cer                                                                -                    GM1             Galβ13GalNAcβ14[NeuAcα23]Galβ14Glc.be                    ta.11Cer                                 -                    Sialylparagloboside                                                                           NeuAcα23Galβ14GlcNAcβ13Galβ14Glc.beta                    .11Cer                                   -                    Sialylparagloboside (NeuGc)                                                                   NeuGcα23Galβ14GlcNAcβ13Galβ14Glc.beta                    .11Cer                                   -                    Sialylneolactofucopenta-                                                                      NeuAcα23Galβ14[Fucα13]GlcNAcβ13Gal.b                    eta.14Glcβ11Cer                     -                    osylcer                                                                       GD1a            NeuAcα23Galβ13GalNAcβ14[NeuAcα23]Gal                    β14Glcβ11Cer                   -                    GD1.sup.b       Galβ13GalNAcβ14[NeuAcα28NeuAcα23]Gal                    β14Glcβ11Cer                   -                    GT1b            NeuAcα23Galβ13GalNacβ14[NeuAcα28NeuA                    cα23]Galβ14Glcβ11Cer     -                    Sialylneolacto- NeuGcα23Galβ14GlcNAcβ13Galβ14GlcNAc.b                    eta.13Galβ14Glcβ11Cer          -                    hexaoslycer                                                                   I-Active Sialyl- lactoisooctaosylcer                                                           ##STR1##                                -                    I-active Lacto- isooctaosylcer                                                                 ##STR2##                                -                    I-active Gal.sub.2 - lactoisooctaosylcer                                                       ##STR3##                                -                    __________________________________________________________________________     .sup.a Trivial names and structures are represented according to              recomendations in ref. 47 and references cited therein; cer, ceramide.        .sup.b Negative binding (-) indicates no binding to 4 μg of lipid and      positive binding to less than 0.5 μg (+++), 0.5 to 2 μg (++), and       2-4 μg (+).                                                           

                  TABLE II                                                        ______________________________________                                        Inhibition of M. pneumoniae adherence to adenocarcinoma                       cell monolayers (WiDr) by dextran sulfate                                     Dextran Sulfate                                                                          .sup.3 H-M. pneumoniae attached (% of control).sup.a               (μg/ml) Exp. 1     Exp. 2     Exp. 3                                       ______________________________________                                         1         69         83         89                                            10        19         77         74                                           100        31(p < 0.002)                                                                            78 (p < 0.05)                                                                            72 (p < 0.1)                                 ______________________________________                                         .sup.a Results are the average of triplicate determinations normalized to     control binding in the absence of inhibitor: 7%, 21%, and 11% of the adde     mycoplasma respectively for the three experiments. Nonspecific binding to     mediumtreated coverslips without cells was 2-3% of the total added. The       significance of the inhibition at 100 μg/ml dextran sulfate, relative      to the control adhesion to WiDr cells in the absence of inhibitor, was        determined using a twosided t test.                                      

Metabolic labelling with [³⁵ S]-sulfate confirmed that WiDr cells make alarge amount of sulfatide, as shown in FIG. 5. An orcinol-positiveresorcinol-negative glycolipid that comigrates with authentic brainsulfatide is detected in acidic lipids from WiDr cells, FIG. 5A, lane c.This lipid contains [³⁵ S], FIG. 5A, lane 1, and was verified to besulfatide by comigration with authentic sulfatide in an acidic solventsystem that resolves sulfatide, seminolipid, and cholesterol-3-sulfate,FIG. 5B, lane a. Based on incorporation of [³⁵ S]under steady statelabelling, WiDr cells contain approximately 90 nmoles sulfatide per gramwet weight of cells. Significant label was also incorporated intocholesterol 3-sulfate and small amounts into a lipid that comigrateswith seminolipid, FIG. 5B, lane a.

To obtain the results shown in FIG. 5, WiDr cells were metabolicallylabelled with [³⁵ S]--sulfate as described above. Neutral and acidiclipids were chromatographed on silica gel high performance thin layerplates developed in chloroform/methanol/0.25% KCl in water, 5:4:1 (PanelA) or chloroform/methanol/acetone/acetic acid/water, 8:2:4:2:1 (panelB). The lipids were detected by autoradiography, lane a, or orcinolreagent, lanes b-f.

In Panel A, [³⁵ S]-labelled acidic lipids from 106WiDr cells, lane a,neutral, lane b, and acidic, lane c, lipids from 30 mg wet weight ofWiDr cells. The orcinol positive sulfatide band is indicated by thearrow (→). Migration of reference glycolipids is indicated in the leftmargin: sulfatide, GM3, GM2, GM1, GD1a, GD1b, and GT1b.

In Panel B, [³⁵ S]-labelled acidic lipids from 106WiDr cells, lanes aand c, bovine brain sulfatide, lane e, and neutral glycolipid standardsfrom top to bottom: CMH, CDH, CTH, and GL4, lane f.

The glycolipid binding specificity of M. pneumoniae was established bythe thin layer overlay assay. Of the many glycolipids present on thechromatogram, as shown in Table I, M. pneumoniae bound only to sulfatedglycolipids and weakly to lactosylceramide, glucosylceramide,lactotrihexaosylceramide, paragloboside, and o-galactosylparagloboside,as shown in FIG. 1. Binding curves of purified sulfatide andlactosylceramide, however, demonstrated that only sulfatide exhibited agood dose response, whereas lactosylceramide bound M. pneumoniae weaklyand the other glycolipids not at all, as shown in FIG. 2. Interestingly,M. pneumoniae does not discriminate between galactosyl ceramide I³-sulfate and its unnatural 6-sulfate isomer, cf. Table 1, yet theorganism does not bind to cholesterol sulfate or to sulfatedglucuronosylparagloboside, which has a terminal sulfate linked to the3-position of glucuronic acid. These results indicate that sulfate aloneis not sufficient for M. pneumoniae binding and that at leastGas(3SO₃)β1-residues in glycolipids are required.

M. pneumoniae did not bind to ═2-3-sialylneolacto-series glycolipidseither on thin layer chromatograms or adsorbed incholesterol-phosphatidylcholine on microtiter plates. This findingappears to be at variance with reports that these glycolipids as well asbrain gangliosides, which lack the noelacto-series core structure,inhibit adhesion of M. pneumoniae, and the finding of the presentinventors that the organism binds to asparagine-linked oligosaccharidesbearing this terminal structure. However, as demonstrated forlaminin-mediated hemagglutination and laminin binding to sulfatide,inhibition by gangliosides may be indirect in that they mask sulfatidereceptors. It is postulated that mannose, which occurs inasparagine-linked glycoproteins but is absent in glycolipids, may berequired for tight binding.

The biological relevance for sulfatide for adhesion of M. pneumoniae issuggested by three findings. First, only metabolically-active M.pneumoniae cells bind to sulfatide, as is shown in FIG. 3. Atphysiological temperatures, binding was maximal in RPMI medium andalmost completely abolished in nutrient-deficient medium or attemperatures of 4° C. These results are consistent with the finding ofothers that adhesion of M. pneumoniae is decreased by metabolic poisons,low temperatures, and by using nonviable organisms, and that adhesionmay be influenced by an energized membrane. Second, sulfatide occurs inhigh amounts in human trachea and is present in human lung and culturedWiDr human colon adenocarcinoma cells, as shown in FIGS. 1 and 5. Thelatter have approximately 50 million sulfatide molecules per cell.Additional sulfated glycoconjugates that are recognized by M. pneumoniaemay be present on glycoproteins or proteoglycans in these tissues.Thirdly, dextran sulfate, which specifically inhibitssulfatide-dependent binding, partially inhibits M. pneumoniae adhesionto WiDr cells.

The existence of receptors other than sialylglycoproteins would explainwhy inhibition of M. pneumoniae binding to cultured cell lines bysialylglycoconjugates or following neuraminidase treatment is usuallyincomplete. This is also the case with inhibition studies in which 1μg/ml dextran sulfate completely abolished binding to purified sulfatideimmobilized on plastic, as shown in FIG. 4, but only partially inhibitedM. pneumoniae adhesion to WiDr cells, cf. Table 2. Thus, there areprobably at least two distinct receptors that mediate binding of M.pneumoniae to cells: glycolipids containing terminalNeuAcα2-2Galβ1-4GlcNAc sequences, both of which must be blocked forcomplete inhibition of M. pneumoniae binding to cultured cells.Sulfatide-mediated adhesion may be important in mycoplasma pathogenicityto guarantee intimate contact of the parasite with the host membrane tosatisfy its strict nutritional requirements.

Adhesion of M. pneumoniae to many cell types in vitro may be mediated byrecognition of sialyloligosaccharides on the host cell surface. Based onselective restoration of binding or beuraminidase-treated erythrocytesusing CMP-sialic acid and purified sialyltransferases, adhesion of theerythrocytes on surface grown sheet cultures of M. pneumoniaespecifically requires sialic acid linked α2-3 to N-acetyllactosaminesequences. Inhibition studies using glycolipids, glycoproteins, andoligosaccharides suggested that sialylated linear or branchedpolylactosamine sequences on both glycoproteins and glycolipids arereceptors an erythrocytes for M. pneumoniae. Although glycolipidsincluding gangliosides inhibited attachment in this and several otheradherence assays, cf. Chandler et al., Infect. Immun. 38: 598-603, 1982;other workers have concluded that binding is mediated by glycoproteinsbut not glycolipids, cf. Gabridge et al., Infect. Immun. 25: 455-459,1979 and Geary et al., Isr. J. Med. Sci. 23: 462-468, 1987. In thelatter study, no sialic acid was found in a purified receptor proteinfrom lung fibroblast (MRC5) cells.

It has now been found that several glycoproteins containing α2-3 but notα2-6 linked sialic acid can support attachment of M. pneumoniae and thatsimple biantennary asparagine-linked oligosaccharides are sufficient toefficiently mediate adhesion. Based on inhibition studies, this bindingspecificity is distinct from sulfatide binding and both mechanisms areinvolved in adhesion to cultured human cells.

M. pneumoniae Adhesion to Immobilized Glycoproteins

Glycoproteins dissolved in 0.01 M sodium phosphate buffer, pH 7.4,containing 150 mM NaCl, 1 mM CaCl₂, and 0.01% NaN₃ were adsorbed ontoplastic polyvinylchloride 86 well microtiter plates by incubation for 16hours at 4° C. The unbound proteins were removed, and the wells werefilled with tris-BSA and incubated for 30 minutes at room temperature.The wells were rinsed with RPMI1640 containing 25 mM HEPES, pH 7.3, and1% bovine serum albumin. M. pneumoniae strain M129 labelled with [³H]-palmitate were dispersed in RPMI-BSA by passing four times through a26 gauge needle, and 50 μl of the suspension was applied to the wells.After incubation for 60 minutes at 37° C., the wells were washed fivetimes with saline and the labelled M. pneumoniae bound to the proteinswere quantified by scintillation counting in Aquasol.

For inhibition studies, sugars in 25 μl of RPMI-BSA were added to wellscoated with 10 μg/ml laminin followed by 25 μl of [³ H]-M. pneumoniae.Binding was determined to both laminin-coated and uncoated wells intriplicate at each inhibitor concentration and in the absence ofinhibitor. In some experiments, the adsorbed proteins were pretreatedwith neuraminidase. After adsorption of the proteins and incubation intris-BSA, the wells were rinsed three times with 50 mM sodium acetate,pH 5.5, containing 150 mM NaCl, 5 mM CaCl₂, 1 mg/ml bovine serumalbumin, and 1 mM phenylmethanesulfonyl fluoride. The wells wereincubated with 0.05 units/ml neuraminidase in the same buffer or withbuffer without enzyme overnight at 20° C. The wells were rinsed threetimes with tris-BSA, and M. pneumoniae binding was determined asdescribed above.

Binding of monoclonal antibody My-28 to the immobilized proteins beforeor after digestion with neuraminidase was determined using a 1:1000dilution of ascites fluid in tris-BSA. After incubation for two hours atroom temperature, the wells were washed three times with tris-BSA. Boundantibody was detected using goat anti-mouse IgM labelled with ¹²⁵ I bythe Iodogen method.

M. pneumoniae Adhesion to WiDr Cells

Adhesion of labelled M. pneumoniae to WiDr cells on glass cover slipswas determined as described previously. For inhibition studies, dextransulfate and 3'-sialyllactose were dissolved in RPMI-BSA and the pH wasadjusted to 7.4 with NaOH. The inhibitors were added to wells containingwashed cover slips with attached WiDr cells or blank cover slipspreincubated in medium or tris-BSA. Labelled M. pneumoniae were addedimmediately and incubated with slow rocking for sixty minutes at 37° C.After the coverslips were washed by dipping six times in saline, boundM. pneumoniae were determined by scintillation counting in Aquasol. InFIG. 6, [³ H]-labelled M. pneumoniae, 630,000 cpm/5×10⁵ CCU, wereincubated in microtiter wells coated in duplicate with laminin ( ○ ) ,fetuin (◯), hCG, (), or transferrin (□) at the indicated concentrations.After washing to remove the unbound organisms, the bound mycoplasma weredetermined by scintillation counting. Binding to uncoated wells was 3%of the applied radioactivity.

Several glycoproteins including laminin, fetuin, and hCG support dosedependent and saturable adhesion of M. pneumoniae when adsorbed onplastic, as shown in FIG. 6. Typically, 20 to 60% of the added M.pneumoniae bound to the wells at saturating protein concentrations.Nonspecific binding to uncoated wells was 0.3 to 3% of the totalradioactivity applied. Binding is energy dependent, and no binding wasdetected in a tris-albumin buffer without glucose. Most proteins,however, are inactive in this assay, as shown in FIG. 6 and Table III.The relative activities of several proteins for promoting M. pneumoniaeadhesion were estimated by comparing the dose response curves, and aresummarized in Table III. The proteins laminin, fetuin, thrombospondin,hCG, and the alpha-subunit of hCG have similar activity and promoteadhesion to wells coated with less than 10 ng of glycoprotein.Glycophorin and alpha-1-acid glycoprotein are weakly active, whereas theother proteins are essentially inactive, promoting binding of less than10% of the added M. pneumoniae at the highest levels tested, 1-5μg/well.

Immunolon 2 microtiter plates and bacteriological polystyrene were alsoexamined as substrates for M. pneumoniae adhesion to adsorbed proteins.Although binding varied with the plastic used, the distinction betweenthe active and inactive glycoproteins was consistently observed with allthree types of plastic. Thus, the differences in activity are probablynot an artifact of selective adsorption of the active glycoproteins.

FIG. 7 illustrates the effect of neuraminidase treatment on M.pneumoniae binding to immobilized glycoproteins. Microtiter wells werecoated with fetuin (circles), hCG (squares), or α-subunit of hCG(triangles) and treated for sixteen hours with 0.05 U/ml neuraminidase(Closed symbols) in sodium acetate buffer, pH 5.5, or buffer alone (opensymbols). [³ H]-labelled M. pneumoniae binding was determined asdescribed above.

Binding to the active glycoproteins requires sialic acid, asneuraminidase treatment of the adsorbed protein as shown in FIG. 7 orpretreatment with neuraminidase in solution before adsorption (resultsnot shown) abolishes all binding activity. Several of the inactiveglycoproteins also contain silaic acid, but the linkage reported inhuman transferrin, fibrinogen, and plasma fibronectin is exclusive α2-6to galactose. The linkage in hCG and a majority of N-linked fetuinoligosaccharides is α2-3. Thus, in agreement with previous studies oferythrocyte adhesion to surface grown sheet cultures of M. pneumoniae,binding of the labelled M. pneumoniae to immobilized glycoproteinsappears to be specific for α2-3-linked sialic acid.

With the exception of hCG, all of the active glycoproteins haveextensive heterogeneity in their carbohydrate structure or have onlypartially characterized structure. HCG contains only mono- andbiantennary asparagine-linked oligosaccharides on both subunits and four0-linked oligosaccharides on the β-subunit. Since the α-subunit of hCGbinds M. pneumoniae as well as the intact protein, as shown in Table IIIand FIG. 7, the O-linked carbohydrates on the β-subunit are not requiredfor binding. Thus, a biantennary asparagine linked carbohydrate withα2-3-linked sialic acid is sufficient for binding of M. pneumoniae.

                  TABLE III                                                       ______________________________________                                        M. pneumoniae binding to glycoproteins                                        adsorbed on plastic                                                           Protein           Relative binding activity.sup.a                             ______________________________________                                        Murine Laminin    1.5                                                         Bovine Fetuin     1.0                                                         hCG               0.7                                                         hCG α-subunit                                                                             0.8                                                         Human platelet thrombospondin                                                                   0.7                                                         Human type MM glycophorin                                                                       0.06                                                        Human α.sub.1 -acid glycoprotein                                                          0.03                                                        Hen Ovomucoid     <0.01                                                       Human Transferrin <0.01                                                       Human plasma Fibrinogen                                                                         <0.01                                                       Human plasma Fibronectin                                                                        <0.01                                                       Bovine serum Albumin                                                                            <0.01                                                       ______________________________________                                         .sup.1 Binding of [.sup.3 H]-M. pneumoniae was determined to polyvinyl        chloride microtiter wells coated with 0.006 to 2 μg of the respective      proteins. Relative binding activities of the proteins were determined by      the amount of protein required to give half maximal binding of mycoplasma     and are expressed relative to fetuin which was assigned a value of 1.0.       Results are the mean values of 2 or 3 experiments.                       

Inhibition of binding after neuraminidase treatment is not due to acontaminating protease as all of the adsorbed proteins bind monoclonalantibody My-28 after neuraminidase treatment. This antibody recognizesN-acetyllactosamine sequences found in glycolipids and glycoproteins. Inmost cases, antibody binding is detected only after neuraminidasedigestion. Excepting glycophorin, which binds about ten-fold lessantibody, all of the asialoglycoproteins have similar binding curveswith antibody My-28. Uniform binding of the antibody to all of theneuraminidase-treated glycoproteins confirms that all of the proteinsare adsorbed on plastic to a similar extent under the conditions used,and that sialylated N-acetyllactosamine sequences on the immobilizedproteins are accessible for binding antibodies and M. pneumoniae.

Binding of M. pneumoniae to adsorbed laminin is inhibited by solublelaminin with 50% inhibition as 80 μg/ml, as shown in Table IV. Bindingis also inhibited by 3'-sialyllactose at comparable concentrations.6'-Sialyllactose is at least ten-fold less active. Neither laminin nor3'-sialyllactose inhibits M. pneumoniae attachment to sulfatide.Conversely, dextran sulfate is a potent inhibitor of binding tosulfatide, but has no effect on attachment on laminin. Thus, the twobinding specifities probably require two independent carbohydratebinding sites on the M. pneumoniae pathogen.

Asparagine-linked oligosaccharides released from fetuin also inhibit M.pneumoniae binding to laminin, as shown in Table IV. Unfractionatedoligosaccharides and the unbound fraction from chromatography onconcanavalin A-Sepharose have similar inhibitory activity to3'-sialyllactose. The latter fraction contains the triantennaryoligosaccharides of fetuin. The bound fraction from the concanavalin-Acolumn, which should contain the biantennary oligosaccharides of fetuin,is about 20-fold more active, inhibiting M. pneumoniae binding by 50% at12 μM.

                  TABLE IV                                                        ______________________________________                                        Inhibition of M. pneumoniae binding to laminin or sulfatide                   adsorbed on plastic                                                                          Substrate                                                      Inhibitor        Laminin     Sulfatide                                        ______________________________________                                                       I.D..sub.50.sup.a                                              3'-sialyllactose 0.3     mM      >5    mM                                     6'-sialyllactose >5      mM.sup.b                                                                              N.D.                                         Fetuin oligosaccharides Con A                                                                  0.3     mM      N.D.                                         unbound                                                                       Fetuin oligosaccharides Con A                                                                  0.012   mM      N.D.                                         bound                                                                         laminin          80      μg/ml                                                                              >200  μg/ml                               dextran sulfate  >200    μg/ml.sup.c                                                                        0.5   μg/ml                               ______________________________________                                         .sup.a Concentration of inhibitor giving 50% inhibition of control            binding. Oligosaccharide concentrations are presented as sialic acid          concentrations determined by the periodateresorcinol assay (12).              .sup.b Binding was 54% of control at 5 mM inhibitor.                          .sup.c Binding was 112% of control at 200 μg/ml dextran sulfate Mr         500,000.                                                                 

FIG. 9 shows the inhibition of M. pneumoniae adhesion to humanadenocarcinoma cell line (WiDr). Adhesion of [³ H]-M. pneumoniae to WiDrcells growing on 13 mm glass cover slips was determined as describedabove. Inhibition by dextran sulfate or 3'-sialyllactose at theindicated concentrations was calculated relative to control bindingdetermined in RPMI/BSA without inhibitors. The results are presented aspercent inhibition (mean±S.D. n=4 with n=8 for determination of controlbinding without inhibitor).

3'-Sialyllactose inhibits M. pneumoniae adhesion to monolayers of WiDrcells. The inhibition is dose dependent, but at concentrations more thanten-fold higher than the ID₅₀ for inhibiting binding to laminin, 40% ofthe control adhesion remains. In the same experiment, dextran sulfatealso gave partial inhibition of M. pneumoniae adhesion. However, whenthe two inhibitors were combined, adhesion was inhibited by 90%. Thus,both binding specificities participate in adhesion to WiDr cells andcomplete inhibition of M. pneumoniae adhesion to these cells can beachieved using a combination of inhibitors for both binding mechanisms.

As can be seen from the above, the adhesive glycoproteins laminin andthrombospondin and several other glycoproteins, when adsorbed onplastic, strongly promote adhesion of M. pneumoniae. The adhesiveactivities of all of these proteins depends on sialic acid on theiroligosaccharides, and is lost after neuraminidase treatment. All or mostof the sialic acid on these proteins is linked α2-3 to galactose. Inhuman plasma fibronectin and fibrinogen and except for a minortriantennary oligosaccharide in transferrin, all sialic acid is linkedα2-6, and no binding of M. pneumoniae was detected. Thus, the specificrequirement for α2-3-linked sialic acid for binding to purifiedglycoproteins is in agreement with previous results for adhesion oferythrocytes to M. pneumoniae.

Whereas the oligosaccharide structures on some of the activeglycoproteins are heterogeneous or only partially characterized, theo-subunit of hCG contains only one monoantennary and one biantennaryasparagine-linked oligosaccharide, and is as active as the otherglycoproteins with more complicated oligosaccharide structures.Therefore, the minimum structure for adhesion of M. pneumoniae isprobably a simple biantennary oligosaccharide with sialic acid linkedα2-3 to the terminal galactoses. Strong inhibition by sialyl biantennaryoligosaccharides from fetuin but not the triantennary oligosaccharidessuggest that this is the preferred structure for M. pneumoniae binding.This is a common structure that is probably present on the surfaceglycoproteins of many cells and could account for the broad range ofcell types that show sialic acid-dependent M. pneumoniae binding.

The same terminal sequence that is found on the asparagine-linkedoligosaccharides of the glycoproteins that bind M. pneumoniae,Sialα2-3Galβl-4GlcNAcβ-, occurs on glycolipids yet does not supportbinding of M. pneumoniae when the glycolipids are immobilized on thinlayer plates or in a phosphatidyl choline/cholesterol monolayer onplastic. The orientation of the sequence may be different in glycolipidsso that it is not recognized or sterically inhibited from binding M.pneumoniae, or additional sugar residues such as the mannose found onlyon the glycoproteins may be required for high avidity binding.

Although some have proposed that polylactosamine sequences are requiredfor M. pneumoniae binding, neither fetuin, thrombospondin, nor hCG haspolylactosamine sequences, although laminin does have these sequences.Furthermore, human α1 acid glyco-protein has polylactosamine sequences,but they are uncommon in glycophorin and neither protein binds M.pneumoniae well. This contrasts with the finding that the latter twoproteins inhibit erythrocyte adhesion to M. pneumoniae better thanfetuin, cf. Loomes et al., Nature (Lond): 307: 560-563, 1984.Glycophorin contains hydrophobic regions; however, inhibition of sialicacid-dependent erythrocyte binding and invasion by merozoites ofPlasmodium falciparum malaria results from a toxicity of the hydrophobicpeptide of glycophorin. A similar toxicity may account for inhibition ofM. pneumoniae adhesion, as a "receptor" for glycophorin was isolatedfrom M. pneumoniae membranes but its binding was inhibited as well by ahydrophobic peptide of glycophorin which lacks carbohydrate as by theintact glycophorin.

The ability of fetuin to bind M. pneumoniae when adsorbed on plastic isinteresting since fetuin is a major protein in fetal calf serum, whichis a component of the growth medium for most of the cell types that havebeen used for attachment assays. Thus, fetuin may adsorb onto the glasssubstrates or onto the surface of WiDr and other cells and account forall or part of the sialic acid-dependent adhesion. "Non-specific"adhesion of M. pneumoniae to coverslips preincubated in mediumcontaining fetal calf serum is specifically inhibited by3'-sialyllactose but not by dextran sulfate. 3'-Sialyllactose, however,does not inhibit nonspecific binding to coverslips preincubated intris-BSA.

The inhibition studies tabulated in Table IV indicate that M. pneumoniaehas two distinct adhesins that recognize sulfated glycolipids andα2-3-linked sialyl oligosaccharides on glycoproteins, respectively.Based on the complete dependence on erythrocyte sialyloligosaccharidesfor binding, only the latter receptor is required for bindingerythrocytes. Inhibition of M. pneumoniae adhesion to cultured celllines by an inhibitor of sulfatide binding or following neuraminidasetreatment, however, is usually incomplete. As shown in FIG. 9, theeffects of 3'-sialyllactose and dextran sulfate are additive and nearlycomplete inhibition is obtained with both inhibitors, suggesting thatboth types of carbohydrates are utilized by M. pneumoniae to adhere tothese cells in vitro. Based on these results, it is unlikely that agentsinhibiting binding to either carbohydrate receptor could preventinfection by blocking adhesion to host epithelium, but a combination ofthe two types of inhibitors may prevent infection by M. pneumoniae.

Mycoplasma pneumoniae have been found to bind specifically toglycolipids that contain sulfated esters of galactose, particularly assulfatide and lactosylsulfatide, and this binding can be specificallyinhibited in vitro by dextran sulfate. Thus, dextran sulfate or theGal(SO₄)β1-sequence immobilized onto insoluble carriers or supports canbe used in agglutination or enzyme-linked assays to specifically detectM. pneumoniae or M. hominus in body fluids or other solution. TheGal(3SO₄)β1-sequence or dextran sulfate can be used to remove M.pneumoniae or M. hominus from body fluids or other fluids, and toaffinity purify components on the bacterial cell that mediate attachmentto host tissue in the infection process.

In order to treat a patient infected with Mycoplasma hominus orMycoplasma pneumoniae, the dextran sulfate or compound carrying the SO₃--Gal-β-1-Ceramide sequence is combined with a pharmaceuticallyacceptable carrier and administered to a patient in amount sufficient tobind the pathogen and remove it from the system of the patient. Theseamounts, which are readily determined by those skilled in the art, canrange from approximately 0.1 gram to about 5 grams per patient per dayuntil there is evidence of successful treatment of the infection.

Compositions within the scope of the invention include compositionswherein the active ingredient is contained in an effective amount toachieve its intended purpose. Determination of the effective amount is,of course, within the skill in the art.

In addition to the active Mycoplasma binding compound, thepharmaceutical compositions according to the present invention maycontain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activeingredients into preparations which can be used pharmaceutically totreat infection with the Mycoplasma pathogens.

Preferably, the preparations, particularly those which can beadministered orally and which can be used for the preferred type ofadministration, such as tablets, dragees, and capsules, and preparationswhich can be administered rectally, such as suppositories, as well assuitable solutions for administration orally or by injection, containfrom about 0.1 to about 99 percent, and preferably from about 25-85percent, of active ingredient, together with the excipient.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, such as by means ofconventional mixing, granulating, dragee-making, dissolving orlyophilizing. The pharmaceutical preparations for oral use can beobtained by combining the active ingredients with solid excipients andprocessing the compounds, after adding suitable auxiliaries, if desiredor necessary, to obtain tablets or dragee cores.

Suitable excipients include fillers such as sugars, for example,lactose, sucrose, mannitol, or sorbitol, cellulose preparations and/orcalcium phosphates, such as tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste using starches suchas maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylcellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone. If desired,disintegrating agents may be added, such as the above-mentioned starchesand carboxymethyl starch, crosslinked polyvinyl pyrrolidone, agar,alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above, all, flow-regulating agents and lubricants, such as silica,talc, stearic acid or salts thereof, and/or polyethylene glycol. Drageecores are provided with suitable coatings which, of desired, areresistant to gastric juices. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures. Inorder to produce coatings resistant to gastric juices, solutions ofsuitable cellulose preparations such as acetyl-cellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize different combinations ofactive compound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of the activecompounds in a suppository base. Suitable suppository bases are, forexample natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. In addition, it is alsopossible to use gelatin rectal capsules which consists of a combinationof the active compounds with a base. Possible base materials include,for example, liquid triglycerides, polyethylene glycols, or paraffinhydrocarbons.

Suitable formulations for parenteral administration and irrigation ofdiseased tissues include aqueous solutions of the active ingredients inwater-soluble form. In addition, suspensions of the active ingredientsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension containsstabilizers.

The manner in which detection methods are conducted using the receptorsof the present invention will be readily appreciated by persons skilledin the art. A protein which contains sialyloligosaccharide receptors,such as fetuin, is immobilized onto a solid carrier, such as polystyrenebeads. Any Mycoplasma hominus or Mycoplasma pneumoniae present in a testfluid will bind with the sialyloligosaccharide receptors on the carrier.The presence of the Mycoplasma is then detected by an enzyme-dextransulfate conjugate in a rapid ELISA technique, or by latex agglutination.

Compositions prepared according to the present invention are used totreat patients infected with one of the Mycoplasma pathogens, such as M.pneumoniae or M. hominus. A composition according to the presentinvention is administered to the patient for the duration of appearanceof symptoms of infection. This time period is determined without undueexperimentation by one skilled in the art. Determination of infectioncan be accomplished by detecting the pathogens in sample of bodily fluidfrom the patient or from examining the clinical symptoms of infection,such as fever, shivering, etc.

Diseased tissue can be irrigated with a liquid composition containingthe dextran sulfate or compound containing the SO₃ --β1-1Gal sequence toremove any Mycoplasma pathogens from the tissue. Preferably, a dilutesolution containing approximately 0.1 to 5 grams per liter of the activeingredient is contained in a pharmaceutically acceptable carrier, suchas saline solution. Irrigation of the tissue is continued until thereappear to be no further pathogens present in the tissue.

The dextran sulfate or other SO₃ -β1-1Gal-sequence containing compoundmay alternatively be immobilized onto an insoluble carrier, such aspolystyrene beads, glass cover plates, or the like, and used inagglutination or enzyme-linked assays to detect M. pneumoniae or M.hominus in body fluids or other solutions.

Additionally, dextran sulfate or other compound containing the SO₃ -β₁-lGal-sequence may be immobilized on a suitable carrier and used toremove these bacteria from body fluids so as not to interfere with otherassays. The compound is adsorbed onto a carrier and introduced into thefluid, whereby the M. pneumoniae or M. hominus adhere to the carrier andthe carrier containing the bacteria is removed from solution.

While the invention is described above in relation to certain specificembodiments, it will be understood that many variations are possible,and that alternative materials and reagents can be used withoutdeparting from the invention. In some cases such variations andsubstitutions may require some experimentation, but such will onlyinvolve routine testing.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology herein is for the purpose of description and not oflimitation.

what is claimed is:
 1. A receptor which is capable of binding toMycoplasma hominus and Mycoplsma pneumoniae comprising a compoundselected from the group consisting of compounds containing the structureSO₃ ⁻ -Galβ1-1Ceramide.
 2. A receptor according to claim 1 wherein saidcompound containing the structure SO₃ ⁻ -Galβ1-1Ceramide is selectedfrom the group consisting of sulfatides and sialyloligosaccharides.
 3. Apharmaceutical composition comprising the receptor according to claim 1in a pharmaceutically acceptable carrier.
 4. The composition accordingto claim 3 wherein said pharmaceutically acceptable carrier is solublein water.
 5. The composition according to claim 3 wherein saidpharmaceutically acceptable carrier is insoluble in water.
 6. A methodfor removing a pathogen selected from the group consisting of Mycoplasmahominus and Mycoplasma pneumoniae from a sample, comprising contactingsaid sample with a receptor according to claim 1, incubating said samplewith said receptor for a sufficient period of time to bind said sampleto said receptor, and removing said receptor from said sample.
 7. Themethod according to claim 6 wherein said receptor is carried on a solid,water-insoluble carrier.
 8. A method for treating a patient infectedwith a pathogen selected from the group consisting of Mycoplasma hominusand Mycoplasma pneumoniae comprising administering to said patient aneffective amount of a composition according to claim
 3. 9. A method fordetecting a pathogen selected from the group consisting of Mycoplasmapneumoniae and Mycoplasma hominus comprising:immobilizing fetuin onto asuitable carrier; contacting a sample to be analyzed with theimmobilized fetuin; and detecting the presence of the pathogen using anenzyme-dextran sulfate conjugate.
 10. A method of preventing infectionin a host by a pathogen selected from the group consisting of Mycoplasmapneumoniae and Mycoplasma hominus comprising administering to a host aneffective amount of a composition according to claim
 3. 11. A method fortreating diseased tissues which is infected with a pathogen selectedfrom the group consisting of Mycoplasma pneumoniae and Mycoplasmahominus comprising contacting said diseased tissues with a compositionaccording to claim 3.